The present invention relates to modified dimeric nucleoside compounds, oligonucleotide analogs prepared therefrom and methods of their use. In one aspect of the present invention, oligonucleotide analogs are provided that contain modified nucleoside dimers that enhance the hybridization of the oligonucleotide analogs to, for example, RNA. More specific objectives and advantages of the invention will hereinafter be made clear or become apparent to those skilled in the art during the course of explanation of preferred embodiments of the invention.
It is well known that most of the bodily states in mammals, including most disease states, are affected by proteins. Classical therapeutic modes have generally focussed on interactions with such proteins in an effort to moderate their disease-causing or disease-potentiating functions. However, recently, attempts have been made to moderate the actual production of such proteins by interactions with molecules that direct their synthesis, such as intracellular RNA. By interfering with the production of proteins, maximum therapeutic effect and minimal side effects may be realized. It is the general object of such therapeutic approaches to interfere with or otherwise modulate gene expression leading to undesired protein formation.
One method for inhibiting specific gene expression is the use of oligonucleotides and oligonucleotide analogs. Certain oligonucleotide analogs have been accepted as therapeutic agents with great promise. Oligonucleotides and oligonucleotide analogs are known to hybridize to single-stranded DNA or RNA molecules. Hybridization is the sequence-specific base pair hydrogen bonding of nucleobases of the oligonucleotide or the oligonucleotide analog to the nucleobases of the target DNA or RNA molecule. Such nucleobase pairs are said to be complementary to one another.
In determining the extent of hybridization to a complementary nucleic acid, the relative ability of an oligonucleotide or an oligonucleotide analog to bind to the complementary nucleic acid may be compared by determining the melting temperature of a particular hybridization complex. The melting temperature (Tm), a characteristic physical property of double helices, denotes the temperature (in degrees centigrade) at which 50% helical (hybridized) versus coil (unhybridized) forms are present. Tm is measured by using the UV spectrum to determine the formation and breakdown (melting) of the hybridization complex. Base stacking, which occurs during hybridization, is accompanied by a reduction in UV absorption (hypochromicity). Consequently, a reduction in UV absorption indicates a higher Tm. The higher the Tm, the greater the strength of the bonds between the strands.
For use as therapeutics, oligonucleotides and oligonucleotide analogs must be transported across cell membranes or be taken up by cells, and appropriately hybridize to target DNA or RNA. These critical functions depend on the initial stability of the oligonucleotides toward nuclease degradation. A serious deficiency of unmodified oligonucleotides which affects their hybridization potential with target DNA or RNA for therapeutic purposes is the enzymatic degradation of administered oligonucleotides by a variety of intracellular and extracellular ubiquitous nucleolytic enzymes referred to as nucleases. For oligonucleotides to be useful as therapeutics or diagnostics, the oligonucleotides should demonstrate enhanced binding affinity to complementary target nucleic acids, and preferably be reasonably stable to nucleases and resist degradation. For a non-cellular use such as a research reagent, oligonucleotides need not necessarily possess nuclease stability.
A number of chemical modifications have been introduced into oligonucleotides to increase their binding affinity to target DNA or RNA and resist nuclease degradation. The present invention describes the use of oligonucleotide analogs having modified dimers. These modified dimers have unexpectedly enhanced binding affinity when placed in an oligonucleotide.
While it has been recognized that nucleosides and oligonucleotides bearing base and sugar modifications are useful, there remains a long-felt need for oligonucleotides with greater binding affinity, hence improved hybridization characteristics, and greater nuclease resistance. Such oligonucleotides are desired as therapeutics, diagnostics, and research reagents.
The present invention presents modified dimeric nucleoside compounds having Formula I: 
wherein
Z is a covalent intersugar linkage;
each T1 and T2 is, independently, xe2x80x94OH, xe2x80x94OR, xe2x80x94CH2R, xe2x80x94NH(R), xe2x80x94SH, xe2x80x94SR, or a protected hydroxyl;
BX is a heterocyclic base;
X is F, Oxe2x80x94R, Sxe2x80x94R or Nxe2x80x94R(R2);
R is alkyl, or a ring system having from about 4 to about 7 carbon atoms or having from about 3 to about 6 carbon atoms and 1 or 2 hetero atoms wherein said hetero atoms are selected from oxygen, nitrogen and sulfur and wherein said ring system is aliphatic, unsaturated aliphatic, aromatic or heterocyclic;
and wherein any available hydrogen atom of said ring system is each replaceable with an alkoxy, alkylamino, urea or alkylurea group;
or R has one of the formulas: 
xe2x80x83wherein
Q is O, S or NR2;
m is from 1 to 10;
y is from 0 to 10;
E is N(R2)(R3), Nxe2x95x90C(R2)(R3), C1-C10 alkyl, or C1-C10 substituted alkyl wherein said substituent is N(R2)(R3);
each R2 and R3 is, independently, H, C1-C10 alkyl, alkylthioalkyl, a nitrogen protecting group, or R2 and R3, together, are a nitrogen protecting group or wherein R2 and R3 are joined in a ring structure that can include at least one heteroatom selected from N and O; and R1 is H or C1-C12 alkyl.
In further preferred embodiments of the compounds of the invention, oligonucleotide analogs are provided comprising at least one moiety having the Formula II: 
wherein:
each Z is a covalent intersugar linkage;
T3 is a nucleotide other than a ribonucleotide, a nucleoside other than a ribonucleoside, hydroxyl, a blocked hydroxyl, or an oligonucleotide wherein the 3xe2x80x2-terminal nucleotide of said oligonucleotide is not a ribonucleotide.
T4 is a nucleotide, a nucleoside, an oligonucleotide, a hydroxyl or a blocked hydroxyl;
with the proviso that at least one of said T3 and T4 is not a hydroxyl, or blocked hydroxyl;
BX is a heterocyclic base;
each X is, independently, F, xe2x80x94Oxe2x80x94R, xe2x80x94Sxe2x80x94R, or xe2x80x94Nxe2x80x94R(R2);
R is alkyl, or a ring system having from about 4 to about 7 carbon atoms or having from about 3 to about 6 carbon atoms and 1 or 2 hetero atoms wherein said hetero atoms are selected from oxygen, nitrogen and sulfur and wherein said ring system is aliphatic, unsaturated aliphatic, aromatic or heterocyclic;
and wherein any available hydrogen atom of said ring system is each replaceable with an alkoxy, alkylamino, urea or alkylurea group;
or R has one of the formulas: 
xe2x80x83wherein
Q is O, S or NR2;
m is from 1 to 10;
y is from 0 to 10;
E is N(R2)(R3), Nxe2x95x90C(R2)(R3), C1-C10 alkyl, or C1-C10 substituted alkyl wherein said substituent is N(R2)(R3); and
each R2 and R3 is, independently, H, C1-C10 alkyl, alkylthioalkyl, a nitrogen protecting group, or R2 and R3, together, are a nitrogen protecting group or wherein R2 and R3 are joined in a ring structure that can include at least one heteroatom selected from N and O; and R1 is H or C1-C12 alkyl.
In some preferred embodiments, compounds are provided that contain at least one moiety of Formula III: 
wherein:
Z is a covalent intersugar linkage;
BX is a heterocyclic base;
X is F, xe2x80x94Oxe2x80x94R, xe2x80x94Sxe2x80x94R or xe2x80x94NR(R2);
R is alkyl, or a ring system having from about 4 to about 7 carbon atoms or having from about 3 to about 6 carbon atoms and 1 or 2 hetero atoms wherein said hetero atoms are selected from oxygen, nitrogen and sulfur and wherein said ring system is aliphatic, unsaturated aliphatic, aromatic or heterocyclic;
and wherein any available hydrogen atom of said ring system is each replaceable with an alkoxy, alkylamino, urea or alkylurea group;
or R has one of the formulas: 
xe2x80x83wherein
Q is O, S or NR2;
m is from 1 to 10;
y is from 0 to 10;
E is N(R2)(R3), Nxe2x95x90C(R2)(R3), C1-C10 alkyl, or C1-C10 substituted alkyl wherein said substituent is N(R2)(R3);
each R2 and R3 is, independently, H, C1-C10 alkyl, alkylthioalkyl, a nitrogen protecting group, or R2 and R3, together, are a nitrogen protecting group or wherein R2 and R3 are joined in a ring structure that can include at least one heteroatom selected from N and O;
T3 is a nucleotide other than a ribonucleotide, a nucleoside other than a ribonucleoside, a hydroxyl, a blocked hydroxyl, or an oligonucleotide wherein the 3xe2x80x2-terminal nucleotide of said oligonucleotide is not a ribonucleotide; and
R1 is H or C1-C12 alkyl.
In some preferred embodiments, oligonucleotide analogs of the invention are provided that contain a plurality of moieties of Formulas II or III.
In some preferred embodiments of the foregoing compounds, X is xe2x80x94Oxe2x80x94R. In other preferred embodiments X is xe2x80x94Oxe2x80x94R and R is xe2x80x94CH3.
In further preferred embodiments of the compounds of the invention, R is xe2x80x94CH2xe2x80x94CH2xe2x80x94Oxe2x80x94CH3. In still further preferred embodiments of the compounds of the invention, Z is xe2x80x94N(CH3)xe2x80x94Oxe2x80x94CH2xe2x80x94.
In preferred embodiments, oligonucleotide analogs of the invention are prepared to have a predetermined length. In some preferred embodiments, the length is from 1 to 200 subunits. In further preferred embodiments, the length is from 10 to 25 subunits, with from 12 to 20 subunits being more preferred.